Tumor Antigens and T-Cell Antigen Recognition Preclinical as well as clinical studies have shown that anti-tumor T cells are critical for tumor eradication. As a result, a major area of cancer research is the development of vaccines capable of targeting a wide range of cancers. T cells recognize peptides derived from proteins within cells that are presented on the surface of cells in complex with HLA molecules. Our studies focus on identifying tumor peptides that could be used in vaccines targeting carcinomas, and on a novel type of peptide construct for cancer vaccines. In addition, we are investigating the mechanisms involved in T cell recognition of HLA /peptide complexes leading to T cell activation and function. Current evidence indicates that CD4+ T helper cells (Th cells), as well as CD8+ cytotoxic T lymphocytes (CTL) are required for effective anti-tumor immunity, and tumor peptide-based vaccines are more effective when they include tumor specific Th cell- and CTL-defined peptides. Recently, we have focused our efforts on the use of p53-derived peptides as good candidates for the development of broadly applicable vaccines. Ideally, a broadly applicable p53-based vaccine should incorporate both a Th cell-defined wild type (wt) p53 peptide and CTL-defined wt p53 peptides. While four HLA-A2-restricted, CTL-defined wt p53 epitopes are ready for incorporation into vaccines, only two p53 helper peptides are known. Both are HLA-DR4-restricted, with the average frequency of expression of this allele among the population being less than 25%. We set out, therefore, to identify a multiple HLA-DR-binding wt p53 helper peptide using an in vitro strategy applied to enriched populations of CD4+ T cells isolated from PBMC of HLA-DR4 normal donors. These experiments resulted in the identification of the wt p5325-35 peptide as a naturally presented, Th cell-defined peptide restricted by HLA-DR-7 and HLA-DR-11 alleles. The average frequencies of expression of these alleles among the population are 14% and 15.5%, respectively, comparable to that of the HLA-DR4 allele, which is 24.4%. Consequently, the identification of the wt p5325-35 and p53110-124 peptides increases the percentage of HLA-A2+ subjects eligible to receive a multi-epitope p53 peptide-based vaccine incorporating a p53 helper peptide to more than 50%. In previous work, we explored the use of a novel type of multi-epitope peptide vaccine for the induction of anti-tumor CTL. These vaccines, called Trojan Peptides consist of several CTL epitopes joined to each other by furin-sensitive linkers and are attached to either the carboxyl or amino terminus end to a short peptide sequence derived from HIV-tat, which serves as a Protein Transduction Domain (PTD). Our published results showed that these constructs not only penetrate into the cytoplasm, but find their way to the ER and Golgi in a TAP-independent manner, where furin helps to process them into CTL epitopes. We are continuing to evaluate the use of Trojan Peptides as vaccines for cancer. We are now utilizing a mouse breast cancer model based on transgenic expression of the activated rat HER2/neu gene. These mice develop spontaneous breast tumors at a young age (18-22 weeks). We will evaluate the use of these vaccines for the treatment and prevention of breast tumors and determine whether the addition of T helper cell epitopes (MHC class II) will enhance their immunogenicity and capacity to generate immune memory.The majority of T cell-defined tumor peptides is derived from non-mutated "self" antigens and yield weak responses. In many instances, optimized peptides are used to induce T cells that cross-react with the "self" peptides. The underlying mechanism(s) that led to T cell activation are also reflected in the "antigenic mimicry" between viral antigens and "self" which can be the cause of deleterious autoimmune responses.